This invention relates to antibody-drug conjugates of tubulysin analogs having enhanced stability, tubulysin analog-linker compounds for making such antibody-drug conjugates, methods for preparing such antibody-drug conjugates and for their use.
A type of anticancer agent that is generating strong interest is an antibody-drug conjugate (ADC, also referred to as an immunoconjugate). In an ADC, a therapeutic agent, also referred to as the drug, cytotoxin, payload, or warhead, is covalently linked to an antibody whose antigen is expressed by a cancer cell (tumor associated antigen). The moiety covalently linking the antibody and the drug is referred to as the linker. In the case where each antibody has one drug attached to it, the structure of an ADC can be generally represented as:[Antibody]-[Linker]-[Drug]
The antibody, by binding to the antigen, delivers the ADC to the cancer site. There, cleavage of the linker or degradation of the antibody leads to the release of the therapeutic agent. Conversely, while the ADC is circulating in the blood system, the therapeutic agent is held inactive because of its covalent linkage to the antibody. Thus, the therapeutic agent used in an ADC can be much more potent (i.e., cytotoxic) than ordinary chemotherapy agents because of its localized release. For a review on ADCs, see Schrama et al. 2006. (The full bibliographic citation for this and other documents cited herein by first author or inventor and year are listed at the end of this specification.)
One class of compounds that has been proposed as the drug in an ADC are tubulysin analogs. The tubulysins are anti-mitotic naturally occurring cytotoxins, first isolated from myxobacteria cultures. During mitosis, a cell's microtubules reorganize to form the mitotic spindle, a process requiring the rapid assembly and disassembly of microtubules from their constituent proteins α- and β-tubulin. The cytotoxicity of the tubulysins derives from their ability to prevent the assembly of the tubulins into microtubules, causing the affected cells to accumulate in the G2/M phase and undergo apoptosis (Khalil et al. 2006).
The tubulysins have a tetrapeptidyl scaffold consisting of one proteinogenic and three non-proteinogenic amino acid subunits as shown in formula (A): N-methylpipecolinic acid (Mep), isoleucine (Ile), tubuvaline (Tuv), and either tubuphenylalanine (Tup, R′ equals H) or tubutyrosine (Tut, R′ equals OH). Structural variations among the tubulysins (named A, B, etc.) center around residues R′, R″ and R′″ of formula (A), as shown in Table I.

TABLE INaturally Occurring TubulysinsTubulysinR′R″R′″AOHOC(═O)MeCH2OC(═O)i-BuBOHOC(═O)MeCH2OC(═O)n-PrCOHOC(═O)MeCH2OC(═O)EtDHOC(═O)MeCH2OC(═O)i-BuEHOC(═O)MeCH2OC(═O)n-PrFHOC(═O)MeCH2OC(═O)EtGOHOC(═O)MeCH2OC(═O)CH═CH2HHOC(═O)MeCH2OC(═O)MeIOHOC(═O)MeCH2OC(═O)MeUHOC(═O)MeHVHOHHYOHOC(═O)MeHZOHOHHPretubulysinHHMe
Cheng et al. 2013 disclose ADCs of tubulysin analogs, in particular analogs having at the R′ position of formula (A) above an amino (NH2) group, which can serve as an attachment site for the linker.
The acetate group in the Tuv subunit appears to be essential for biological activity. Its removal (deacetylation), resulting in compounds in which R″ in formula (A) is hydroxyl, reportedly leads to loss of biological activity (Domling et al. 2006). In a study of tubulysins U and V, which differ in the former being acetylated and the latter being deacetylated, tubulysin V was reported to be less potent by about 200× to 600×, depending on the assay (Balasubramanian et al. 2009). Because an acetate group can be susceptible to hydrolysis, deacetylation at the R″ position is a concern in the development of tubulysin analogs as the drug in an ADC. If deacetylation occurs, cleavage of the linker would lead to release of an inactive drug.
Cong et al. 2015 have proposed addressing this issue by replacing the naturally occurring acetate group in the Tuv subunit with a more hydrolytically resistant moiety such as a carbamate:

However, in a field as complex as the development of pharmaceuticals, a tubulysin analog having carbamate group in the Tuv subunit will not necessarily function identically to an analog having the naturally occurring acetate group in each and every instance. Therefore, it is desirable to develop a solution to the issue of acetate hydrolysis, in which the acetate group is preserved, as an alternative for those instances in which the pharmaceutical properties of the carbamate group are not entirely coincident with those of the acetate group.